By Jennifer Elizabeth Grant, Hong Li
This quantity highlights proteomics experiences of quantitative PTM alterations in either peripheral and crucial worried approach proteomes using the newest advances in mass spectrometry. Chapters contain useful details touching on the basics of pattern instruction, liquid chromatography, and tandem mass spectrometry instrumental research and should elucidate top practices within the interpretation of information utilizing smooth bioinformatics ways. Written for the preferred Neuromethods series, chapters comprise the type of aspect and key implementation suggestion that guarantees profitable ends up in the laboratory.
Authoritative and practical, Analysis of Post-Translational changes and Proteolysis in Neuroscience aims to determine profitable leads to the additional research of this very important field.
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Extra resources for Analysis of Post-Translational Modifications and Proteolysis in Neuroscience
Again make sure to remove air bubbles from the narrow inlet of the column before doing so. Do not apply vacuum (as advised against by the manufacturer). 5. 1 % TFA). 6. 1 % TFA. 7. Place columns above new 15 or 50 mL polypropylene tubes to collect eluate. 1 % TFA, 40 % acetonitrile). 8. Freeze the eluate on dry ice (or À80 C freezer) for 2 h to overnight and lyophilize frozen peptide solution for a minimum of 2 days to assure that TFA has been removed from the peptide sample. 4. NOTE: The lyophilization should be performed in a standard lyophilization apparatus.
Transfer the eluate into a new tube. Wash the beads twice with 200 μL elution buffer, then combine the resulting solutions with the eluate 8. 6 PNGase F Treatment 1. Dissolve PNGase F in heavy-oxygen water to a concentration of 1 U/μL 2. Dissolve lyophilized glycopeptide (entirely dry) in 100 μL of heavy-oxygen water 3. Add 5 μL of 1 U/μL PNGase F 4. 7 1. 4 % (vol/vol). 0; otherwise add more 10 % TFA 2. 4) 3. 6 Â 250 mm 5 μm particle reversed phase column. Set up the HPLC gradient method as follows (Table 1) 4.
14 Hongbo Gu et al. 4. Homogenize the tissue sample using a Polytron set at maximum speed: 2 Â 20-s pulses. Chill on ice for 1 min between each pulse. 5. Sonicate the tissue homogenate. First cool on ice for about 1 min, and then sonicate using a microtip set to 15 W output with three bursts of 30 s each. Cool on ice for 1 min between each burst. 6. Centrifuge the cell homogenate/lysate to clear cell debris. Centrifuge at 20,000 rcf (g) at 4 C for 15 min, and then transfer the supernatant (this is your protein sample) to a 50 mL screw-cap bottle.
Analysis of Post-Translational Modifications and Proteolysis in Neuroscience by Jennifer Elizabeth Grant, Hong Li
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